Immunostimulatory dna:rna oligonucleotides

Abstract

Immunostimulatory sequence-specific RNA oligonucleotides corresponding to 3′ terminal sequences of single-stranded minus-sense RNA genomic RNAs are provided. Also provided are compositions and methods relating to an immunostimulatory 4-mer RNA motif provided as 5′-C/U-U-G/U-U-3′. Incorporation of this short RNA motif is sufficient to confer new and altered immunostimulatory properties in new and existing oligonucleotides, including CpG oligodeoxynucleotides. Also provided are methods for use of the immunostimulatory RNA oligonucleotides and DNA:RNA chimeric oligonucleotides of the invention to induce an immune response in vitro and in vivo, as well as to treat allergy, asthma, infection, and cancer in a subject. Single-stranded oligoribonucleotides of the invention are believed to signal through a Toll-like receptor (TLR) chosen from TLR9, TLR8, TLR7, and TLR3. The oligoribonucleotides can also be used in a method to screen for TLR antagonists.

1 - 36 . (canceled) 
     
     
         37 . An immunostimulatory composition comprising a chimeric DNA:RNA oligonucleotide 7-40 nucleotides long comprising 
       
         
           
                 
                 
               
                     
                   5′-dX 1 -N 1 -C/U-U-G/U-U-N 2 -dX 2 -3′ 
                 
             
                
               
            
           
         
       
       wherein U is uracil (U) oxyribonucleoside, C/U is cytosine (C) oxyribonucleoside or uracil (U) oxyribonucleoside, G/U is guanine (G) oxyribonucleoside or uracil (U) oxyribonucleoside, dX 1  and dX 2  independently are DNA sequences 0-6 nucleotides long wherein at least one of dX 1  and dX 2  is at least 1 nucleotide long, and N 1  and N 2  independently are RNA sequences 0-10 nucleotides long. 
     
     
         38 . The chimeric oligonucleotide of  claim 37 , wherein N 1  and N 2  are both 0 nucleotides long. 
     
     
         39 . The immunostimulatory composition of  claim 37 , wherein dX 1  is 0 nucleotides long. 
     
     
         40 . The immunostimulatory composition of  claim 37 , wherein dX 2  is 0 nucleotides long. 
     
     
         41 . The immunostimulatory composition of  claim 37 , wherein dX 1 , dX 2 , or both dX 1  and dX 2  comprise a CpG motif. 
     
     
         42 . The immunostimulatory composition of  claim 37 , further comprising at least one stabilized internucleotide linkage. 
     
     
         43 . The immunostimulatory composition of  claim 37 , further comprising at least one phosphorothioate internucleotide linkage. 
     
     
         44 . The immunostimulatory composition of  claim 37 , wherein 5′-C/U-U-G/U-3′ is 5′-CUGU-3′. 
     
     
         45 . The immunostimulatory composition of  claim 37 , wherein 5′-C/U-U-G/U-3′ is 5′-UUGU-3′. 
     
     
         46 . The immunostimulatory composition of  claim 37 , wherein 5′-C/U-U-G/U-3′ is 5′-CUUU-3′. 
     
     
         47 . The immunostimulatory composition of  claim 37 , wherein 5′-C/U-U-G/U-3′ is 5′-UUUU-3′. 
     
     
         48 . (canceled) 
     
     
         49 . The immunostimulatory composition of  claim 37 , wherein the chimeric DNA:RNA oligonucleotide is associated with a cationic lipid. 
     
     
         50 . The immunostimulatory composition of  claim 37 , further comprising an antigen. 
     
     
         51 - 103 . (canceled)